PROJECT SUMMARY Transmitochondrial cytoplasmic hybrid (cybrid) cells, in which cells contain the same nucleus but different mitochondrial genomes has have led to the striking observation that mitochondrial DNA (mtDNA) could influence cancer metastasis. This observation has been corroborated by a number of groups though reports conflict as to the nature of mtDNA able to influence metastatic behavior of cancer cells. Initial reports identified the ability of severe missense mutations in protein encoding regions of the mitochondrial genome to promote metastasis while others have claimed that minor missense mutations can also have this effect and are actually more potent and promoting metastasis. Others have demonstrated that mutations in non-protein coding regions of the mitochondrial genome and even synonymous mtDNA variants could also influence the metastatic behavior of cancer cells. The relevance of these observations to human disease is supported by evidence that mtDNA mutations are widely present in clinical samples and that certain mutations significantly associate with worse clinical outcomes. Further emphasizing the ability of mtDNA to influence tumor progression and metastasis, clinical studies have identified single nucleotide polymorphisms and mtDNA macro-haplotypes that confer significant increased risk of metastasis and relapse even when adjusted for clinical and pathological co-variables. Despite these robust and reproducible observations spanning from experimental model systems to clinical cohorts of cancer patients, there is little consensus as to the nature of mtDNA species able to influence the metastasis of cancer cells. Further, the mechanism by which mtDNA influences metastatic cell behavior is unclear. Preliminary data from our lab suggest that no common mtDNA mutation identifies metastatic cells; rather the metastatic potential of several ROS-generating mtDNA mutations is largely determined by their surrounding mtDNA genomic landscapes, which can act as enhancers or repressors of metastasis. The mtDNA landscapes of metastatic cells are characterized by activation of the SIRT3 axis of the mitochondrial unfolded protein response (UPRmt). We found that heterogeneous activation of this pathway is seen within primary tumors breast cancer patients and that patient-matched metastatic lesions have significantly increased activation of this pathway, suggesting a selection for cells which activate the SIRT3 axis of the UPRmt during the metastatic cascade. The proposed studies in this application will build upon this preliminary data to further characterize the role of the SIRT3 axis of the UPRmt in metastatic progression and mechanistically understand the role that mtDNA plays in activating this pathway. Our published preliminary data, in addition to other literature in the field, suggest that activation of the UPRmt may mechanistically underlie the reported ability of mtDNA to influence cancer cell metastasis. The innovative studies proposed in this application will provide critical insight into our understanding of cancer and mitochondrial biology and may identify novel targets for the treatment of metastatic disease.